Detection of nitric oxide by electron paramagnetic resonance spectroscopy

Abstract

Electron paramagnetic resonance (EPR) spectroscopy has been used in a number of ways to study nitric oxide chemistry and biology. As an intrinsically stable and relatively unreactive diatomic free radical, the challenges of detecting this species by EPR are somewhat different from those of transient radical species. This review gives a basic introduction to EPR spectroscopy and discusses its uses to assess and quantify nitric oxide formation in biological systems.

Figure 1. Single-line EPR spectrum displayed as both a first derivative (upper) and absoption spectrum (lower)

Spectra are simulations created using WinSim.

Figure 2. The effect of magnetic nuclei on the EPR spectrum showing the hyperfine interaction

A) The effect of nucleus with nuclear Spin of one half. B) The effect of a nucleus with a nuclear spin of 1. Spectra are simulations created using WinSim.

Figure 3. Nitronyl nitroxides as NO detectors/scavengers

A) Reaction of nitronyl nitroxide with NO showing conversion to an immino nitroxide and nitrogen dioxide. B) Typical EPR spectral changes that occur upon reaction of a nitronyl nitroxide with NO.

Figure 4. The Iron dithiocarbamate/NO complex, and the structure of the more water soluble ligand MGD

Figure 5. EPR spectra of metHb and HbNO

A) MetHb was prepared by incubating human oxyHb with ferricyanide followed by purification on sephadex G25 size exclusion chromatography. The metHb was placed in an EPR tube frozen. The EPR spectrum was taken at 5K using the following EPR parameters. B) EPR spectra of HbNO. EPR spectra of HbNO consist of a superposition of at least three EPR spectra. These derive from the β-chain heme iron-NO complex, the α-chain heme iron NO complex in a 6-coordinate geometry (T-state) and the α-chain heme iron complex in a 5-coordinate geometry (R-State). (Adapted from a Figure originally published in The Journal Of Biological Chemistry Piknova, B., Gladwin, M.T., Schechter, A. N., and Hogg, Nl. Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation. J. Biol. Chem. 280, 40583-40588. 2005. © the American Society for Biochemistry and Molecular Biology.

Figure 6. Dinitrosyl Iron complexes in RAW264.7 macrophages

RAW264.7 mouse macrophage-like cells were cultured to confluence. Either spermineNONOate or S-nitrosocysteine were added and incubated for 30 minutes. Cells were scraped and placed in an EPR tube and frozen. EPR spectra were taken at 77K.